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dc.contributor.authorPETRUZZELLI, LILLI MARY
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 48-06, Section: B, page: 1619.
dc.description.abstractInsulin activates a tyrosine-specific protein kinase in lectin-enriched extracts from both 3T3-L1 adipocytes and human placenta. The protein kinase activity copurifies with the insulin binding activity to homogeneity. The procedure used to purify the receptor from placenta included membrane solubilization, chromatography on agarose-bound wheat germ agglutinin and affinity chromatography on insulin-Sepharose. Elution of the receptor from insulin-Sepharose was performed with 0.5 M NaCl and 1 mM dithiothreitol at pH = 5.5. The addition of DTT was essential for full recovery of the protein kinase activity. It was concluded that the insulin binding and insulin-dependent protein tyrosine kinase are intrinsic components of the same oligomer since (1) they copurify to homogeneity, (2) are immunoprecipitated by antibodies to the receptor, and (3) phosphorylation occurs by an intramolecular reaction. Molecular cloning and expression of the human insulin receptor cDNA has confirmed the fact that the insulin receptor is a protein tyrosine kinase.;A similar receptor that has a high molecular weight (350,000-400,000) and contains a subunit that is crosslinked to 125-labelled insulin has been identified in Drosophila melanogaster. Although adult Drosophila melanogaster contain protein tyrosine kinase activity, insulin-dependent phosphorylation has only been detected in Drosophila melanogaster embryos (0-21h). The kinase activity appears at 6-12 hours of embryogenesis, increases during the 12-18 hour period and falls to low levels in adults. A genomic fragment of DNA that is 53% identical to the protein tyrosine kinase domain of the human insulin receptor was isolated from Drosophila melanogaster. An antipeptide antibody directed against a sequence in the kinase domain of the human insulin receptor and that is conserved in Drosophila melanogaster immunoprecipitates a 95,000-dalton species that is phosphorylated in tyrosine residues. A single 11kb polyA{dollar}\sp+{dollar}-selected mRNA hybridizes to the clone and its expression parallels the appearance of the tyrosine protein kinase activity. These results suggest that the fragment encodes the insulin receptor and that it may play a role in embryogenesis in Drosophila.
dc.publisherProQuest Dissertations & Theses

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